Phenylalanine Residues in the Active Site of Tyrosine Hydroxylase: Mutagenesis of Phe300 and Phe309 to Alanine and Metal Ion-Catalyzed Hydroxylation of Phe300
详细信息 查看全文
- 作者：Holly R. Ellis ; S. Colette Daubner ; Ruth I. McCulloch ; and Paul F. Fitzpatrick
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 24, 1999
- 年：1999
- 卷：38
- 期：34
- 页码：10909 - 10914
- 全文大小：93K
- 年卷期：v.38,no.34(August 24, 1999)
- ISSN：1520-4995
文摘
Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in therecently described three-dimensional structure of the enzyme, where they have been proposed to playroles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated dueto a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C.(1998) Biochemistry 37, 13437-13445]. Mutants of tyrosine hydroxylase with alanine substituted forPhe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% lessactivity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the productionof dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPAand dihydropterin. The K6-MPH4 value for F300A tyrosine hydroxylase is twice the wild-type value. Theseresults are consistent with Phe309 having a role in maintaining the integrity of the active site, whilePhe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolatedcatalytic domain after incubation with a large excess of 7,8-dihydropterin, DTT, and Fe2+. The modificationis not observed in the untreated catalytic domain or in the full-length protein, even in the presence ofexcess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallographyconditions and is not relevant to catalysis.